Financial support:
This work was supported by a grant (FAIR CT 97-3097) from the European
Community.
Keywords: biological activity, cyclic AMP, peptone, radioimmunoassay,
radioreceptorassay.

Abstract

Calcitonin gene related peptide (CGRP)
related molecules were purified from sardine hydrolysates prepared
using 0.1% alcalase and two hours of hydrolysis. Gel exclusion chromatography
and HPLC performed purification of these molecules. The purified molecules
were characterised using specific CGRP radioimmunoassays and radioreceptoraasays.
From 22 mg of crude extract, we obtained 14 µg of CGRP related molecules,
the molecular weight determined by mass spectrophotometry was 6000
daltons. The biological activity of these molecules was analysed using
the ability of CGRP to stimulate the adenylate cyclase activity in
rat liver membranes. The purified molecules induced an inhibition
of the CGRP stimulated adenylate cyclase activity, this effect was
specific as no such effect was observed on the glucagon stimulated
adenylate cyclase activity measured in the same rat liver membrane
preparation. These results suggest that the purified molecules may
act as antagonists for peptides that bind to CGRP receptors in rat
liver membranes. These new antagonists may be of particular importance
in various aspects of CGRP action in vertebrates.

Article

Many synthetic compounds as well as almost all organisms including
animal, plants and microorganisms have been screened for bioactive
substances as starting materials for biomedicines. Although numerous
substances have been isolated, there are probably many others, especially
degradation products of proteins that have not been identified. Through
a control of process parameters such as pH, time and enzyme-substrate
ratio, it is possible to produce hydrolysates whose components may
have retained various functional properties attached to the native
molecules (Adler-Nissen, 1982). Previous experiments
performed using a large range of hydrolysates from various sources
demonstrate the presence of CGRP immunorelated molecules in various
fish hydrolysates (Fouchereau-Peronet al. 1999).
The calcitonin gene related peptide (CGRP), a 37 aminoacid neuropeptide,
is derived from the same gene as calcitonin by a mechanism of alternative
splicing (Amaraet al. 1982). It is predominantly
synthesised in neural tissue and is mainly involved in the control
of vasodilatation, with inotropic and chronotropic effects on the
heart (Franco-Cerecedaet al. 1987) but is
also implicated in the regulation of gastric acid secretion (Hugheset al. 1984; Lenzet al. 1984). This
peptide can also inhibit the proliferative response of T lymphocytes
to mitogens (Umeda and Arizawa, 1989) and macrophage
activation (Nonget al. 1989). In addition,
at high doses, CGRP induces the same effects as calcitonin, that is
hypocalcemia and hypophosphatemia (Rooset al.
1986). In non mammalian vertebrates, this peptide is mainly found
in gills and intestine (Fouchereau-Peronet al.
1990b) and is involved in the control of hydromineral metabolism
by its specific action on gill membranes (Fouchereau-Peronet al. 1990a; Arlot-Bonnemainset al.1991). The sequence similarity between calcitonin and CGRP
suggests that both peptides may support identical biological effects
mainly in the control of homeostasis and reproduction (Lopez
et al. 1976; Bjornsson et al. 1986).

Among the various tested
hydrolysates, the sardine (Sardina pilchardus) hydrolysates
were characterised by the highest quantity of CGRP immunologically
and biologically related molecules (Fouchereau-Peron
et al. 1999). In addition, we demonstrated that using increasing
hydrolysis time and various alcalase concentrations, the CGRP like
molecules were mainly found after 2 hours of hydrolysis using an alcalase
concentration of 0.1% (Ravallec-Plé et al. 2001).
Therefore, sardine hydrolysates were prepared using these conditions
and purified by gel exclusion and HPLC chromatography. The purified
CGRP like molecules were then analysed for their CGRP like biological
effect using the ability of CGRP to stimulate the adenylate cyclase
activity in rat liver membranes.

Sardine hydrolysates
were prepared from cooked head and guts using 0.1% alcalase 2.4L (Novo
Nordisk Industri DK-2880 Bagsvaerd, Denmark) in phosphate buffer (0.1
M, pH 8.1, 1/10, w/v). Hydrolysis was carried out during 2 hours at
40°C. The enzyme was inactivated by 15 min of boiling in a microwave
oven. After centrifugation at 25000 g, 20 min, the supernatant was
filtrated through a polyethylene net, ultrafiltrated (cut off 10 KD)
and freeze dried.

Radioimmunoassays.
Immunoreactive CGRP was measured following a previously described
assay for human CGRPI (Fouchereau-Peron et al. 1990b):
in brief, an anti-CGRP antiserum at a final dilution of 1/250,000
was incubated with serial dilutions of synthetic human CGRP or sardine
hydrolysates. Antiserum was first incubated with tissue extracts or
standard peptide for 18 h at 30°C, then 125I labelled human
CGRP was added and the incubation continued during 24 hours at 4°C.
Bound and free hormone was separated by charcoal- dextran precipitation.
Control (specific antibody omitted) tubes were incubated in each assay.
The hydrolysate was assayed in duplicate at multiple dilutions.

Liver
membrane preparation. Liver membranes were obtained from male
Wistar rats and prepared according to the method of Neville until
step 11 (Neville, 1968). Proteins were quantified
by the method of Lowry using BSA as standard (Lowryet al. 1951).

Radioreceptorassays.
Receptor binding ability of immunoreactive molecules was developed
using rat liver membranes and 125I labelled human CGRP.
Incubations, in a 400 µl final volume, were performed at 22°C during
1 hour (Yamaguchiet al. 1988a). At the end
of the incubation, bound and free ligand was separated by centrifugation
in a solution containing 10% sucrose. Data were expressed as specific
binding that was obtained by subtracting from the total binding the
amount of radioactivity associated to the membranes in the presence
of 2 µM CGRP. Receptor binding ability of each purified fraction was
determined in triplicate and expressed as the quantity of protein
(µg) that induced a 25% inhibition of the initial binding.

Positive fractions were
then subjected to HPLC on a C18 prosphere column using a linear gradient
of 10 to 60% acetonitrile in 0.1% TFA. The biological activity of
these fractions was measured by radioreceptorassay and positive fractions
were then repurified using the same column and a linear gradient of
30 to 60% acetonitrile in 0.1% TFA. Optical density was measured at
226 nm.

Protein
determination. The protein concentration of each analysed fraction
was performed using the bicinchoninic acid method using BSA as the
standard (Smithet al. 1985).

Results

Purification
of sardine hydrolysates

Sardine hydrolysate
(22 mg of protein suspended in 7 ml of eluant) was loaded on the HW40
toyopearl column. 2 ml fractions were collected and a 0.5 ml aliquot
was used to quantify the CGRP immunoreactivity. The CGRP immunoreactive
profiles of this preparation (Figure 1) demonstrated
5 main immunoreactive fractions (referred as A, B, C, D and E). These
fractions were further analysed using the CGRP radioreceptorassay.
Only the second tested fraction was able to inhibit the labelled CGRP
binding as the unlabeled hormone did (data not shown). Fraction B
was further purified by HPLC chromatography (Figure
2). 250 µg of proteins were loaded on the column, 1 ml fractions
were collected and a 0.4 ml aliquot used to determine the CGRP immunoreactivity.
Immunoreactive profile of this separation gave three main immunoreactive
peaks. These immunoreactive fractions were tested for their ability
to inhibit the 125I CGRP binding to rat liver membranes.
The data showed that the most efficient inhibiting effect resulted
with the immunoreactive material present in the third peak. Fifty
percent inhibition was observed with 13 µg of proteins from this peak.
This fraction eluted for an acetonitrile concentration of 35%. In
the same conditions, human CGRP eluted for 33.5% acetonitrile.

53 µg proteins of this
fraction were subjected to a second HPLC chromatography using the
same column and a linear acetonitrile gradient from 30 to 60%. Two
absorbency peaks were observed (Figure 3): Only
the fraction 9 showed CGRP -like immunoreactivity. In the CGRP radioreceptorassay,
this fraction displaced the labelled CGRP binding: 25% inhibition
was observed with 370 ng of proteins, that is a K0.5 of
6.5 x 106M-1. No significant displacement was
observed with the second peak. So, from 22 mg of sardine hydrolysate
proteins (Table 1) we obtained 14 µg of a molecule
that present CGRP biological and immunological activity. The purification
factor obtained was about 12.500. The different purification steps
were followed using the CGRP radioreceptorassay. While 311 µg of proteins
from crude extract were necessary to displace 50% of the initial CGRP
binding to rat liver membranes, only 0.9 µg of proteins from the final
purification step produced the same inhibitory effect (Table
1). The molecular weight of this molecule determined by mass spectrophotometry
was 6000 daltons.

Adenylate
cyclase activity of the purified fraction

The biological activity
of the fraction obtained from the second HPLC was analysed by referring
to the capacity of CGRP to stimulate the adenylate cyclase activity
in rat liver membranes. The effect of this fraction was analysed on
the CGRP stimulated adenylate cyclase activity (Figure
4). Increasing protein concentrations between 0.15 and 1.2 µg
of proteins induced a dose related inhibition of the stimulated activity.
25% inhibition was observed with 0.3 µg of proteins, that is a K0.5
of 106M-1. In order to analyse the specificity
of the observed inhibition, we compared the effect of the fraction
purified after gel exclusion chromatography on the CGRP (4 ng/ml)-
and glucagon (200 ng/ml) stimulated adenylate cyclase activity in
the same rat liver membrane preparation (Figure
5). The used glucagon concentration represents the dose inducing
the maximal response in rat liver membranes (Hanoune
et al. 1977). At these concentrations, the stimulated adenylate
cyclase activity represented a two and twenty fold increase over the
control (8.4 ± 0.4 and 11 ± 1 pmoles of cyclic AMP per min
and per mg of proteins) for CGRP and glucagon, respectively. The inhibiting
effect of the prepurified fraction was significant only on the CGRP
stimulated adenylate cyclase activity. In the presence of 1.1 µg of
proteins, the inhibiting effect observed was 14 and 35% of the glucagon
and CGRP stimulated adenylate cyclase activity, respectively. The
same protein concentration induced a 10% inhibition of the liver membrane
adenylate cyclase activity.

Discussion

Many bioactive peptides
have been isolated from enzymatic hydrolysates of proteins (Hsueh
and Moskowitz, 1973; Piotet al. 1992;
Nakagomiet al. 2000). Indeed, invertebrate
and fish by-products might be the best source for such active biopeptides
because these substances would make good starting materials for safer
and less toxic medicines. In addition, these bioactive peptides may
be used as adjuvants to stimulate food intake and to enhance growth
and disease resistance of animals

We reported here the
purification from sardine hydrolysates of CGRP related molecules.
Sardine hydrolysates prepared using specific conditions, that is two
hours of hydrolysis with 0.1% alcalase were subjected to purification
by size exclusion chromatography and HPLC. The appearance of the CGRP
related molecules was followed using both radioimmunoassays and radioreceptorassays.
From 22 mg of proteins present in the crude extract we obtained 14
µg of CGRP related molecules. These molecules were obtained with a
purification factor of 12500 and present a molecular weight of 6000
daltons. This molecular weight is identical to the apparent molecular
weight we reported after exclusion chromatography of sardine hydrolysates
prepared using a different condition that is four hours of hydrolysis
with 1% alcalase (Fouchereau-Peron et al. 1999).

In order to analyse
the function of these molecules, we tested their ability to modulate
the adenylate cyclase activity in rat liver membranes: a specific
target tissue for CGRP. These experiments show that they have a specific
inhibitory action on the CGRP stimulated adenylate cyclase activity.
No such inhibitory effect was observed on the glucagon stimulated
adenylate cyclase activity of the same membrane preparation. The same
results were observed using hydrolysates prepared using different
conditions that is after 4 hours of hydrolysis with 0.1% alcalase
(data not shown). So, whatever the conditions of hydrolysis, the use
of alcalase allows to obtain CGRP related molecules whose molecular
weight is around 6000 daltons and that present an inhibitory action
on the CGRP induced adenylate cyclase activity in rat liver membranes.

So these purified molecules
are able to decrease the 125I labelled CGRP binding to
rat liver membranes and to inhibit the following biological effect.
This action both at the receptor and the adenylate cyclase level is
similar to that observed using a well known competitive inhibitor
of CGRP action: the human CGRP 8-37 (Chibaet
al. 1989). 0.3 µg of our purified proteins, that is 1 10-6M,
induced a 25% inhibition of the CGRP stimulated adenylate cyclase
activity. In the same conditions and using the same rat liver membrane
preparation, the effect of 10-6M CGRP 8-37 induced the
same inhibiting effect. Thus, our purified CGRP related proteins appear
at least as efficient as CGRP8-37 a specific antagonist of CGRP action
in rat liver membranes. So, these purified molecules may act as antagonists
for peptides that bind to CGRP receptors in rat liver membranes. In
addition, they are probably C terminal fragments as all CGRP antagonists
which were reported belong to this class of molecules (Ristet al. 1998).

So, the enzymatic hydrolysis
of sardine by-products allows obtaining molecules immunologically
and biologically related to CGRP. The presence of these molecules
in fish hydrolysates is not surprising as immunoreactive CGRP like
molecules are mainly found in gill and intestine of non mammalian
vertebrates (Fouchereau-Peron et al. 1990b). In non-mammalian
vertebrates, this peptide, by its action on the gill, controls hydromineral
metabolism. In addition, the high circulating levels of CGRP in teleosts
suggests that this neuropeptide is indispensable for everyday functions
such as food intake in teleosts. Recent data demostrated a direct
relationship between the CT and calcium plasma levels in eels after
a high calcium diet (Suzuki et al. 1999).

The development of new
antagonists may be of particular importance in various aspects of
the CGRP action mainly in the control of feeding where CGRP together
with amylin exert a direct or indirect control of meal size and the
control of meal initiation (Geary, 1999).

References

Adler-Nissen, J. (1982). Limited enzymic degradation
of proteins: A new approach in the industrial application of hydrolases.
Journal of Chemical Technology and Biotechnology 32:138-156.